Abstract
High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y-scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y-positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20–100 nm/pixel), complicating single-molecule analysis. Thus, AFM systems able to image large sample areas at high speeds and with nanometer resolution have still been missing. Here, we describe a HS-AFM sample-scanner system able to record large topographic images (≤ 36 × 36 µm2) containing up to 16 megapixels, providing molecular resolution throughout the image frame. Despite its large size, the flexure-based scanner features a high resonance frequency (> 2 kHz) and delivers stable operation even at high scans speeds of up to 7.2 mm/s, minimizing the time required for recording megapixel scans. We furthermore demonstrate that operating this high-speed scanner in time-lapse mode can simultaneously identify areas of spontaneous 2-dimensional Annexin A5 crystal growth, resolve the angular orientation of large crystalline domains, and even detect rare crystal lattice defects, all without changing scan frame size or resolution. Dynamic processes first identified from overview scans can then be further imaged at increased frame rates in reduced scan areas after switching to conventional HS-AFM scanning. The added ability to collect large-area, high-resolution images of complex samples within biological-relevant time frames extends the capabilities of HS-AFM from single-molecule imaging to the study of large dynamic molecular arrays. Moreover, large-area HS-AFM scanning can generate detailed structural data sets from a single scan, aiding the quantitative analysis of structurally heterogenous samples, including cellular surfaces.
Highlights
High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules
Advancements in electronics, miniaturization of cantilevers, and improvement in piezoelectric scanner design have contributed to the continuous development of high-speed atomic force microscopy (HS-AFM) over the past two decades[1]
In this paper we report the adaptation and optimization of a wide-range x, y, z-nano-positioner design previously developed for high-speed scanning ion conductance microscopy (SICM) imaging[12,16] for HS-AFM scanning
Summary
High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. To achieve wide-area imaging without sacrificing frequency bandwidth, wide high-speed scanner designs deploying mechanical amplification of the piezo-driven extension system were introduced[9,10] In such scanners, large x- and y-travel ranges > 40 μm were achieved by mechanically amplifying the displacement of small x- and y-piezo actuators using a third-class leverage mechanism and parallel-kinematic scanner a rrangement[10]. Recent further improvement on this basic design led to the development of a flexure-based, parallelkinematic x, y, z-nano-positioner for scanning ion conductance microscopy (SICM)[12] This scanner achieves a large x, y-travel range of ~ 34 μm, high stage resonance frequency of ~ 2.3 kHz, and minimal cross-coupling between x- and y-axes[13]. SICM imaging at up to 3.5 s/frame over a scan area of 25 × 25 μm[2] was demonstrated, but a similar design has not been incorporated into an AFM scanner
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
